Method of increasing recovery of heterologous active enzymes produced in plants

ABSTRACT

A method of increasing recovery of active enzyme produced in a plant is provided where the enzyme requires a transitional metal cofactor for activation. The metal cofactor is supplied to the enzyme during plant development, during extraction, or after extraction. Recovery of active enzyme is also provided by incubating the extracted enzyme at a non-enzyme degrading temperature. Addition of a negative ion salt further improves active enzyme recovery. Optimum salt concentrations for recovery of laccase from plants using copper solutions is provided.

[0001] This application is a continuation-in-part of previously filedand co-pending application U.S. Ser. No. 60/211,732, filed Jun. 15,2000, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Plants as biofactories for the production of proteins is atechnology that is being employed by a number of groups for ediblevaccines, pharmaceuticals and industrial enzymes (Hood and Jilka, 1999;Hood and Howard, 1999). Pharmaceutical and vaccine production in plantshas several advantages in that the material contains no contaminatingorganisms and can be directly consumed (Hood and Jilka, 1999; Hood andHoward, 1999). Production of industrial enzymes in plants provides thepossibility of considerably reduced production costs, the benefit ofrecovered costs through sale of by products, easier transportation andreduced chance of contamination.

[0003] Over-expression of an industrial enzyme in a transgenic plantrequires quite high expression levels to make the system economicallyviable, a condition that has been achieved for several proteins, e.g.the diagnostic protein, avidin (Hood et al. 1997) and laccase (WO00/20615). Using plants as biofactories for industrial enzyme productionprovides considerable advantages over traditional methods of such enzymeproduction, since plants provide easier transport and cost savings, butalso can be far more readily produced in large quantities than whenproduced in bacteria or fungi, for example, allowing for even furtherincreases in the amount of enzyme which may be produced.

[0004] Achieving high levels of enzyme production in plants is impactedby several factors, such as location of expression of the enzyme withinspecific tissues and within particular subcellular compartments toinsulate the plant tissues from the activity of the enzymes. Thus, in WO00/20615, it is discussed that preferentially directing expression tothe seed of the plant and also to plant cell wall tissue and to theendoplasmic reticulum of the plant cell is advantageous in increasingenzyme protein production.

[0005] In addition to increased concentrations of enzymes, it is desiredthat the enzymes exhibit high activity. While some enzymes depend foractivity only on their structure as proteins, others also require one ormore non-protein components, termed cofactors. The cofactors may be ametal ion or an organic molecule called a coenzyme and some enzymesrequire both. Cofactors are generally stable to heat, where most enzymeproteins lose activity on heating. The term holoenzyme is used to referto the catalytically active enzyme-cofactor complex. When the cofactoris absent, the protein, which is catalytically inactive by itself, iscalled an apoenzyme. Transitional metal ions are important cofactors inenzymatic transformation of nonmineral substances in anabolic andcatabolic processes within plant cells. Therefore, the presence of suchtransitional metal ions may be important in providing an active enzyme.

[0006] Plants produce many of these cofactors as an essential element oftheir vegetative growth process in considerable amounts. Thus, one wouldpresume that the plant would supply adequate quantities of the metal ionneeded to produce active enzyme. For example, about four atoms of copperare needed for each molecule of laccase in order to produce activelaccase enzyme. A person skilled in the art would expect there would bemore than enough copper available since there is a considerable amountof copper for enzyme uptake in the plant. In fact, there is about 20 ppmcopper in normal corn tissues, which would be sufficient to supportlaccase accumulation at much greater than 5ng/mg seed weight (see Table1). TABLE 1 Copper Requirements for Laccase Produced in Corn Seed. pgCopper/mg Corn Seed (ppb) ng Laccase/mg Corn Seed (ppm) Required 5 18 50180 200 720

[0007] Thus there is about a thousand times more copper in the cornplant than is necessary to support laccase expression at 5 ng/mg. Thereshould be more than enough available for production of active laccasewhen it is produced in a plant. Instead, the inventors have found thisis not the situation. Unless such transition metals are added over andabove what is pesent in plants, the amount of active enzyme is reduced.By providing such cofactors during plant development and/or during orafter protein extraction from the plant tissue, the amount of activeenzyme is increased, at times greater than ten fold. This isparticularly surprising, since attempts to add the metal cofactor copperto laccase fungal expression systems have not met with success inimproving activation levels of the enzyme.

[0008] Additionally, the inventors have found that by incubating themetal and enzyme while controlling the temperature during incubation,either during extraction or after, it increases the recovery of activeprotein by such possible mechanisms as refolding and stabilization ofthe protein or reoxidation of the transition metal. Negative salt ionsadded during or after extraction of the enzyme with the metal furtheraid in improving recovery of active enzyme.

[0009] Optimal conditions have also been discovered by the inventors forimproved recovery of laccase using the copper cofactor.

SUMMARY OF THE INVENTION

[0010] The invention relates to the discovery by the inventors thatwhile transgenic plants expressing enzymes contain considerablequantities of transitional metal cofactors needed for certain enzymeactivation, it is necessary to provide additional metal ions in order toincrease recovery of active enzyme from plants.

[0011] Therefore it is an object of the invention to provide a processfor increasing recovery of active enzyme from a plant where that enzymerequires a transitional metal cofactor, by providing additional metalcofactor to the enzyme, either during plant development, duringextraction of the enzyme from the plant, following extraction of theenzyme from the plant, or during all three phases.

[0012] A further object of the invention is to increase recovery ofactive enzyme from a plant in which a transitional metal cofactor hasbeen added by further adding a negative salt ion.

[0013] Yet another object of the invention is increasing recovery ofactive laccase which is produced by a plant having a nucleotide sequenceencoding laccase by providing additional copper to such laccase enzymes.

[0014] An object of the invention is a method of increasing recovery ofactive laccase which is produced in a plant having a nucleotide sequenceencoding laccase by adding a negative salt ion to the laccase enzyme,preferably where the ion is chloride.

[0015] A further object of the invention is a method of increasingrecovery of active organophosphate hydrolase which is produced by aplant having a nucleotide sequence encoding organophosphate hydrolase byproviding additional transitional metals such as zinc, nickel, cobalt ormanganese to such organophosphate hydrolase enzymes.

[0016] An object of the invention is a method of increasing recovery ofactive ogranophosphate hydrolase enzymes by adding a negative salt ionto the enzyme, preferably where the ion is chloride.

[0017] The invention further has as an objective incubating the metaland enzyme while controlling temperature of the incubation. Thetemperature that provides improved recovery will vary with time ofincubation but practical considerations indicate that recovery isimproved when the incubation with the metal is for up to several weekswhen at 4° C., preferably up to several days when incubated at roomtemperature (20°-27° C.); preferably at room temperature up to 37° C.for about 20 to 60 minutes when a negative salt ion is added; and up tothree hours at 50° C. Still another object of the invention is toprovide for optimal yield of active laccase produced in plants by usinga solution to extract the laccase having a copper salt solution of0.05mM to 1M copper, preferably 1 mM to 100 mM copper, more preferably10 to 30 mM copper

DESCRIPTION OF DRAWINGS

[0018] FIGS. 1A-C sets forth the nucleotide sequences of the Trameteslaccase gene used in the experiments set forth below.

[0019] FIGS. 2A-D is a schematic representation of the process used togenerate laccase and OPH plasmids described.

[0020]FIG. 3 is p7718, a construct containing the laccase gene driven bythe ubiquitin promoter, containing the barley alpha amylase signalsequence and the maize optimized PAT gene as a selectable marker, drivenby the 35 S promoter. It further contains left and right borders of theT-DNA sequences.

[0021]FIG. 4 is p8908, which is the same as 7718, except it substitutesthe globulin promoter for the ubiquitin promoter.

[0022]FIG. 5 is p7017, a construct which is essentially the same asp7718, except that it also contains the KDEL sequence and a fungalsignal sequence.

[0023]FIG. 6 is p7699, which is essentially the same as p7017, exceptthat the fungal signal sequence is not present.

[0024]FIG. 7 shows a Western blot of seed extracts from various plantlines. Positive controls are 10 and 1 ng of laccase purified fromTrametes versicolor produced recombinantly in Aspergillus fermentationbroth (lanes 1 & 2). Lanes contain: control corn seed extract asnegative control, and seed extracts from an LCB line, an LCC line and anLCG line. Each lane was loaded with ˜20 μg total protein except for LCGwhich was loaded at ˜0.5 μg total protein. Molecular weight markers areshown on the left.

[0025]FIG. 8 shows a graph depicting results of timing of copperaddition to extracts of maize flour. Flour was extracted either with SATalone, or SAT +10 mM copper sulfate. The SAT extract was divided andeither assayed directly, or treated with 10 mM copper sulfate thenassayed. Precipitated proteins were removed from the copper sulfatetreated extract. The amount of active laccase protein was determined byenzyme assay.

[0026]FIG. 9 shows a graph depicting results of transition metalactivation of laccase. Total proteins were extracted from LCB flour withSAT and brought to 10 mM of the salt of each transition metal asindicated. Proteins were incubated at 50° C. for 1 hour, centrifuged andlaccase activity determined by enzyme assay.

[0027]FIG. 10 shows a graph depicting results of copper sulfateactivation of laccase at three temperatures over time. An SAT extract ofLCB flour was divided into three fractions, 10 mM copper sulfate addedand the extracts incubated at the indicated temperatures. Samples wereassayed in the activity assay over the course of three hours.

[0028]FIG. 11 shows a graph depicting results of using chloride ions toassist in the recovery of active laccase. 0.5 M Sodium Chloride wasadded to either the SAT extraction buffer or to the laccase extractafter extraction along with 10 mM CuSO₄. Proteins were incubated for 1hour at room temperature, centrifuged and laccase activity determined byenzyme assay.

[0029]FIG. 12 shows a graph depicting results of LCG seed extracted with20 mM sodium acetate, 0.1 M sodium acetate or 0.5 M sodium acetate andincubated with 10 mM CuSO₄. The sample extracted in 20 mM sodium acetatewas incubated with 10 mM CuSO₄ and 0, 0.1 or 0.5 M of various salts forone hour at room temperature.

[0030]FIG. 13 shows a graph depicting results of LCG flour extracted insodium acetate (SA) and incubated with various concentrations of sodiumchloride and copper sulfate for 30 minutes at room temperature.

[0031]FIG. 14 shows a graph depicting results of comparison of use ofchloride versus sulfate salts. LCG flour was extracted in sodium acetateand incubated with and without 0.5 M sodium chloride and with either nocopper or up to 100 mM copper sulfate or no cupric chloride up to 100 mMcupric chloride for one hour at room temperature.

[0032]FIGS. 15A and B shows two graphs.

[0033]FIG. 15A shows LCB flour extracted with sodium acetate andincubated with varying amounts of copper added either with or withoutsodium chloride added. The solid symbols represent the data forincubating at room temperature. The white symbols represent the laccaseactivity when incubated for one hour at 50° C.

[0034]FIG. 15B shows LCG flour extracted and incubated similarly.

[0035] FIGS. 16A-E shows the nucleotide sequences of the Stachybotrysgene used in the experiments set forth below.

[0036]FIGS. 17A and B is a schematic representation of the process usedto generate plasmid 8971.

[0037]FIG. 18 shows a graph depicting results of LSC seed extracted inSA and copper treated with 10 mM CuSO4 at either 50° C. or ˜25° C.Aliquots were removed at 0, 5, 10, 15, 30, 60 and 120 minutes andcentrifuged to remove the precipitate. Samples were analyzed by enzymeassay and compared to a standard curve purified Stachybotrys laccase.

[0038]FIG. 19 is p8971 which is a construct containing a ubiquitinpromoter, the barley alpha amylase signal sequence, the organophosphatehydrolase gene, with the maize optimized pat selectable marker driven bythe 35S promoter.

[0039]FIG. 20 shows the nucleotide sequences of the organophosphatehydrolase gene.

[0040]FIG. 21 shows a graph depicting results of increases in OPHActivity in callus and seed. OPH-expressing corn callus extract (panelA) or T2 seed extract (panel B) were incubated with various transitionmetals at 50° C. for one hour and analyzed for enzyme activity.

[0041]FIG. 22 shows a graph depicting results of increases in OPHActivity With Sodium Chloride. Seed extracts were incubated with 0.5 MNaCl and 10 mM of various transition metal salts, both chloride andsulfate salt types for one hour at 50° C. then analyzed by enzyme assay.

[0042]FIG. 23 shows a graph depicting results of increases in OPHActivity over time and at various temperatures.

[0043]FIG. 24 is a graph showing the results of addition of bicarbonateon activation of OPH in seed.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0044] The following is presented to illustrate the preferredembodiments of the invention. All references are incorporated herein byreference.

[0045] In the present invention, the inventors have discovered thatrecovery of active enzyme can be considerably improved when that enzymeis one which depends for activity on a metal cofactor, by exposure ofthe plant tissue to enzyme cofactors either during plant development orwhen extracting or activating the enzyme or during all three steps. Thisincreases the amount of active as opposed to inactive enzyme that may beobtained through plant production.

[0046] Metal ions may either act as the primary catalytic center, as abridging group to bind substrate and enzyme together by forming acoordination complex, or an agent stabilizing the conformation of theenzyme protein in its catalytically active form. Enzymes which requiremetal ions are called metalloenzymes. The table below lists some ofthose enzymes which require metal ions as cofactors. TABLE 2 Metalcofactor Enzyme Zn²⁻ Alcohol dehydrogenase Carbonic anhydraseCarboxypeptidase Glucose-6-phosphate dehydrogenase Trioesphophatedehydrogenases Phosphodiesterase Mg²⁺ Phosphohydrolases EndonucleasesPhosphotransferases Mn²⁺ Arginase Phosphotransferases Fe²⁺ or Fe³⁺Cytochromes Peroxidases Catalase Ferredoxin Cu¹⁺ (Cu²⁺) TyrosinaseCytochrome oxidase Ascorbic acid oxidase Laccase K¹⁺ Pyruvate kinase(also requires Mg²⁺) Na¹⁺ Plasma membrane ATPase (also requires K¹⁺ andMg²⁺)

[0047] For a thorough discussion of enzymes, coenzymes and theirbackground, see Lehninger, A. Chapter 8 “Enzymes: kinetics andinhibition” Biochemistry, second edition, The Johns Hopkins UniversitySchool of Medicine (1977), Worth Publishers, Inc.

[0048] Transitional metal ions are involved as cofactors in plantprocesses. By transitional metal ions, it is meant those transitionalelements found in Group II of the periodic chart of the elements. Thoseelements known to be critical for growth of multicellular plants whichare transitional metals include, for example, copper, zinc, manganese,iron. See e.g. Chapter 18, D. W. Rains “Mineral Metabolism” PlantBiochemistry, Third Edit. Bonner and Varner, eds., Academic Press(1976). Such transitional metal ions are found abundantly in the soiland in healthy plants. The table below lists several of the transitionalmetals known to be critical for growth of multicellular plants. They areshown in terms of relative numbers present in plants with respect tomolybdenum. TABLE 3 Concentration in dry matter Relative number 10⁻⁶μg/gm of atoms with Element atoms/gm or ppm respect to molybdenumMolybdenum (for 0.001 0.1 1 comparison) Copper 0.1 6 100 Zinc 0.3 20 300Manganese 1.0 50 1000 Iron 2.0 100 2000

[0049] Since there is no short supply of the transitional metals in theenvironment and in the plant, it would be expected that when aheterologous enzymatic protein is produced in the plant, there would beample metal cofactors available to produce active enzyme. However, theinventors have found that the amount of active enzyme produced by theplants was lower than expected.

[0050] Thus, when producing an enzyme in a plant where the enzymerequires a cofactor ordinarily available in a plant, one must makeavailable the metal ion by adding it to the plant while it is growing,such as spraying in the field, or by extracting the enzyme with themetal ion added to the extracting solutions, or exposing the enzymeextracted from the plant to the metal. By indicating that the enzyme isexposed to the metal cofactor, one skilled understands that any mannerof exposing the metal to the enzyme will suffice. For example, theenzyme may come in contact with the metal cofactor indirectly in plantdevelopment, or directly, as in the processing mechanism. The enzyme andmetal may be incubated together, meaning exposed for a select period oftime.

[0051] By way of example, copper ion is added when producing an enzymein a plant where the enzyme requires copper ion as a cofactor. Copperion is commonly found in a group of enzymes in which oxygen is useddirectly in the oxidation of substrate. Such oxidases includetyrosinase, laccase, and ascorbic acid oxidase. An oxidized productrequires addition of ¼ O₂ to the substrate. Copper ion is suggested tomediate these enzyme transformations by undergoing cyclic oxidation andreduction. See Rains, supra and Price, C. A. Molecular Approaches toPlant Physiology McGraw-Hill, New York (1970). In the example set forthbelow, copper ion is added to plant-produced laccase during or afterextraction, and also can be added during tissue culture or fieldspraying. Zinc is a transitional metal commonly associated with auxinand is believed to prevent oxidation of the hormone in plants. Variousenzyme systems are known to require zinc, such as alcohol dehydrogenase,glucose 6-phosphate and trioesphophate dehydrogenases, carbonicanhydrase, carboxypeptidase and phosphodiesterase. Manganese isassociated as having a role in photosynthesis in plants, and is involvedin oxidation-reduction processes and decarboxylation and hydrolysisreactions. It is involved in a number of plant enzyme systems and is acofactor for arginase and phosphotransferases. Iron functions in plantsas both a structural component and as a cofactor of enzymic reactions.Oxidation-reduction reactions are most commonly associated withiron-containing systems. It is also a cofactor in a number of enzymes(see table 2 for examples).

[0052] More than one transitional metal may be a useful metal inincreasing active enzyme recovered. For example, zinc, manganese,cobalt, magnesium and nickel are potentially useful in improvingrecovery of active organophosphate hydrolase. By testing the variousoptions, one can easily determine which metal is the optimal choice inthis process. Using OPH as an example, it is clear that cobalt is thebest metal for recovery of the optimal levels of active enzyme, butmanganese, nickel, and zinc are also effective.

[0053] While the examples are directed to particular enzymes, it isevident that any enzyme produced in a plant by introduction ofheterologous DNA encoding the enzyme, where that enzyme requires atransitional metal cofactor, is encompassed within the scope of theinvention.

[0054] Addition of a negative salt ion to the recovery process, eitherduring extraction or afterwards, may yield further increases in activeenzyme obtained from the plant. The salt ion or metal salt may be addedto the process. Again, which ion provides optimal recovery can bereadily determined in a comparison of different ions used in theprocess. Any negative ion non-toxic to the plant is an option. Among theoptions readily apparent to one skilled in the art are chloride,sulfate, phosphate, carbonate and bicarbonate ions. Some of these ionshave been associated as potential inhibitors of the enzyme activity.Surprisingly, the inventors have found that not only are such ionseffective in aiding enzyme recovery, but these salts can be particularlyeffective. When using an ion that is an inhibitor of the enzyme, it isnecessary to remove it by any one of the methods well known to thoseskilled in the art, such as dilution, column removal or the like (Pohl,T. 1990). When the ion is removed, the metal cofactor remains and theapparent amount of active enzyme is increased. For example, chloride isan inhibitor of laccase activity, but when used as a salt with copper,it considerably improves active laccase recovery from plants (2-5 fold).It is believed the ion acts on the enzyme to allow easier entry of thenecessary transitional metal thereby increasing the amount of activeenzyme produced.

[0055] Additional improvement in yield of active enzyme can be achievedby incubating the metal and enzyme while controlling the temperatureduring incubation. While not wishing to be bound by any theory, it isbelieved the plant produces the protein in a form such that theincubation process facilitates incorporation of the required metal ionsthus forming an active complex and enzyme configuration. For example, asdescribed below, the addition of copper salt to a transgenic plant thatmakes heterologous laccase, during extraction of laccase from the planttissue or after extraction, increases the yield of active laccase. Thisis surprising in light of the lack of success in increasing the yield ofactive laccase by adding copper salt to fermenters with and afterfermentation of laccase-producing fungi.

[0056] The temperature providing improved recovery will vary with timeof incubation but the temperature must be one that does not denature theenzyme during the time it is incubating. Generally, recovery is improvedwhen the temperature is not less than 4° C. in which case the activationwill proceed too slowly, nor more than 60° C., where the protein willbreak down fairly quickly. However, there are practical limitations foran optimal recovery that is not cost prohibitive but is reasonable interms of time for the reaction. Thus, improved active enzyme recoveryover several weeks is possible when the temperature is low, at 4° C. Infact, at this temperature, the metal solution and enzyme can be leftindefinitely, in storage, for example. When room temperature, that isabout 20° C. to 27° C. is used, incubation can result in good activeenzyme levels in as little as a few minutes but can continue for as longas 18 to 24 hours. At 50° C., the enzyme is mostly activated nearly atthe onset of contact, and continues effectively up to three hours.However, when used with a salt ion, a lower temperature is morepreferred, as breakdown of the product can occur too quickly when 50° C.conditions are applied.

[0057] Selecting the optimal transitional metal and concentration, thesalt ion that may be added and its concentration is a matter ofcomparison of the options. The time and temperature preferred will bedetermined by one skilled in the art depending upon economics andpreferred production methods.

[0058] For example, copper was added to increase active laccaseproduction. Copper concentrations ranging from 0.05 mM to 100 mM copperwere used in a first comparison. For LCB, as described in Example 1below, a plant producing lower levels of laccase (about 3 ng/mg totallaccase) the optimal level of copper concentration was about 10 mM. Withhigher levels of laccase expression as with LCG, also described below(about 30 ng/mg total), it is beneficial to use higher concentrations ofcopper. Thus the optimal copper concentration was about 30 mM copper.Chloride was selected as the salt to use with copper in laccase activityincrease after side by side comparisons with other salt ions.

[0059] Time and temperature for incubation can be determined as was donein the experiments below, and, in general, by using temperatures rangingfrom 4° C. up to 50° C. and measuring recovery. Measurements were takenboth with and without use of the chloride ion in the process. Firstmeasurements occurred at five minutes, then at ten minutes, 20 minutes,30 minutes, one hour, three hours, 18 hours, and one week. It was foundthat most of the active laccase was recovered at 50° C. at about fiveminutes and continued up to one hour, or at room temperature in aboutfive minutes to three to four hours. The experiment was repeated at 4°C. At 24 hours active laccase was still being recovered, so the process,while effective, was not as practical for recovery compared to highertemperature exposure. All three experiments were repeated using 10 mMcopper salt, 30 mM copper salt and 100 mM copper salt. Optimal recoveryoccurred using 10 mM copper salt with LCB. When the higher expressingLCG was used, optimal recovery occurred at 50° C. with 30 mM coppersalt, or at room temperature when 0.5 M NaCl was added. In addition tohigher amounts of copper salt, the LCG required the presence of thechloride salt for maximal laccase recovery. When chloride salt was used,preferred temperatures were 18° C. to 37° C. and maximum recovery ofactive laccase occurred by 10 to 60 minutes with one hour selected asmost usable.

[0060] This straightforward experimental process can be used todetermine optimal parameters for each of the enzymes, metals andnegative salt ions described herein.

[0061] Genes which encode enzymes of interest are available to oneskilled in the art and examples are set forth below of sequences forgenes encoding laccases and organophosphate hydrolase. It will beevident to one skilled in the art that any gene which encodes an enzymerequiring a transitional metal cofactor is encompassed within the scopeof the invention.

[0062] The methods available for putting together a gene as describedabove for improved expression described above can differ in detail.However, the methods generally include the designing and synthesis ofoverlapping, complementary synthetic oligonucleotides which are annealedand ligated together to yield a gene with convenient restriction sitesfor cloning. The methods involved are standard methods for a molecularbiologist.

[0063] Once the gene has been isolated which encodes such enzymes, it isplaced into an expression vector by standard methods. The selection ofan appropriate expression vector will depend upon the method ofintroducing the expression vector into host cells. A typical expressionvector contains prokaryotic DNA elements coding for a bacterialreplication origin and an antibiotic resistance gene to provide for thegrowth and selection of the expression vector in the bacterial host; acloning site for insertion of an exogenous DNA sequence, which in thiscontext would code for the enzyme of interest; eukaryotic DNA elementsthat control initiation of transcription of the exogenous gene, such asa promoter; and DNA elements that control the processing of transcripts,such as transcription termination/polyadenylation sequences. It also cancontain such sequences as are needed for the eventual integration of thevector into the plant chromosome.

[0064] In a preferred embodiment, the expression vector also contains agene encoding a selection marker which is functionally linked to apromoter that controls transcription initiation. For a generaldescription of plant expression vectors and reporter genes, see Gruberet al. (1993).

[0065] Promoter elements employed to control expression of the enzymeencoding gene and the selection gene, respectively, can be anyplant-compatible promoter. Those can be plant gene promoters, such as,for example, the ubiquitin promoter, the promoter for the small subunitof ribulose-1,5-bis-phosphate carboxylase, or promoters from thetumor-inducing plasmids from Agrobacterium tumefaciens, such as thenopaline synthase and octopine synthase promoters, or viral promoterssuch as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or thefigwort mosaic virus 35S promoter. See Kay et al. (1987) and Europeanpatent application No. 0 342 926. See international application WO91/19806 for a review of illustrative plant promoters suitably employedin the present invention. The range of available plant compatiblepromoters includes tissue specific and inducible promoters. In oneembodiment of the present invention, the exogenous DNA is under thetranscriptional control of a plant ubiquitin promoter. Plant ubiquitinpromoters are well known in the art, as evidenced by European patentapplication no. 0 342 926.

[0066] Alternatively, a tissue specific promoter can be provided todirect transcription of the DNA preferentially to the seed. One suchpromoter is the globulin promoter. This is the promoter of the maizeglobulin-1 gene, described by Belanger, F. C. and Kriz, A. L. (1991). Italso can be found as accession number L22344 in the Genebank database.Another example is the phaseolin promoter. See, Bustos et al. (1989).

[0067] One option for use of a selective gene is aglufosinate-resistance encoding DNA and in an embodiment can be thephosphinothricin acetyl transferase (“PAT”) or maize optimized PAT gene(Jayne et al, U.S. Pat. No. 6,096,947) under the control of the CaMV 35Spromoter. The gene confers resistance to bialaphos. See, Gordon-Kamm etal. (1990); Uchimiya et al., (1993), and Anzai et al., Mol. Gen. Gen.219:492 (1989).

[0068] It may also be desirable to provide for inclusion of sequences todirect expression of the protein to a particular part of the cell. Avariety of such sequences are known to those skilled in the art. Forexample, if it is preferred that expression be directed to the cellwall, this may be accomplished by use of a signal sequence and one suchsequence is the barley alpha amylase signal sequence, (Rogers, 1985).Another example is the brazil nut protein signal sequence when used incanola or other dicots. Another alternative is to express the enzyme inthe endoplasmic reticulum of the plant cell. This may be accomplished byuse of a localization sequence, such as KDEL. This sequence contains thebinding site for a receptor in the endoplasmic reticulum. Munro, S. andPelham, H. R. B. (1987).

[0069] Obviously, many variations on the promoters, selectable markersand other components of the construct are available to one skilled inthe art.

[0070] In accordance with the present invention, a transgenic plant isproduced that contains a DNA molecule, comprised of elements asdescribed above, integrated into its genome so that the plant expressesa heterologous enzyme-encoding DNA sequence. In order to create such atransgenic plant, the expression vectors containing the gene can beintroduced into protoplasts, into intact tissues, such as immatureembryos and meristems, into callus cultures, or into isolated cells.Preferably, expression vectors are introduced into intact tissues.General methods of culturing plant tissues are provided, for example, byMiki et al, (1993) and by Phillips et al., (1988). The selectable markerincorporated in the DNA molecule allows for selection of transformants.

[0071] Methods for introducing expression vectors into plant tissueavailable to one skilled in the art are varied and will depend on theplant selected. Procedures for transforming a wide variety of plantspecies are well known and described throughout the literature. See, forexample, Miki et al., supra; Klein et al., (1992); and Weisinger et al.,(1988). For example, the DNA construct may be introduced into thegenomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery, Klein et al., (1987);electroporation, Fromm et al., (1985); polyethylene glycol (PEG)precipitation, Paszkowski et al., (1984); direct gene transfer, WO85/01856 and EP No. 0 275 069; in vitro protoplast transformation, U.S.Pat. No. 4,684,611; and microinjection of plant cell protoplasts orembryogenic callus. Crossway, (1985). Co-cultivation of plant tissuewith Agrobacterium tumefaciens is another option, where the DNAconstructs are placed into a binary vector system. Ishida et al.,(1996). The virulence functions of the Agrobacterium tumefaciens hostwill direct the insertion of the construct into the plant cell DNA whenthe cell is infected by the bacteria. See, for example Horsch et al.,(1984), and Fraley et al. (1983).

[0072] Standard methods for transformation of canola are described byMoloney et al., (1989). Corn transformation is described by Fromm et al.(1990) and Gordon-Kamm et al, supra. Agrobacterium is primarily used indicots, but certain monocots such as maize can be transformed byAgrobacterium. U.S. Pat. No. 5,550,318. Rice transformation is describedby Hiei et al., (1994), Christou et al., (1991). Wheat can betransformed by techniques similar to those used for transforming corn orrice. Sorghum transformation is described by Casas et al, supra and byWan et al., (1994). Soybean transformation is described in a number ofpublications, including U.S. Pat. No. 5,015,580.

[0073] In one preferred method, the Agrobacterium transformation methodsof Ishida supra and also described in U.S. Pat. No. 5,591,616, aregenerally followed, with modifications that allow the inventors torecover transformants from HII maize. The Ishida method uses the A188variety of maize that produces Type I callus in culture. In onepreferred embodiment the High II maize line is used which initiates TypeII embryogenic callus in culture. While Ishida recommends selection onphosphinothricin when using the bar or PAT gene for selection, anotherpreferred embodiment provides for use of bialaphos instead.

[0074] The bacterial strain used in the Ishida protocol is LBA4404 withthe 40kb super binary plasmid containing three vir loci from thehypervirulent A281 strain. The plasmid has resistance to tetracycline.The cloning vector cointegrates with the super binary plasmid. Since thecloning vector has an E. coli specific replication origin, it cannotsurvive in Agrobacterium without cointegrating with the super binaryplasmid. Since the LBA4404 strain is not highly virulent, and haslimited application without the super binary plasmid, the inventors havefound in yet another embodiment that the EHA101 strain is preferred. Itis a disarmed helper strain derived from the hypervirulent A281 strain.The cointegrated super binary/cloning vector from the LBA4404 parent isisolated and electroporated into EHA 101, selecting for spectinomycinresistance. The plasmid is isolated to assure that the EHA101 containsthe plasmid.

[0075] Further, the Ishida protocol as described provides for growingfresh culture of the Agrobacterium on plates, scraping the bacteria fromthe plates, and resuspending in the co-culture medium as stated in the'616 patent for incubation with the maize embryos. This medium includes4.3g MS salts, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride,1.0 ml thiamine hydrochloride, casamino acids, 1.5 mg 2,4-D, 68.5gsucrose and 36g glucose, all at a pH of 5.8.In a further preferredmethod, the bacteria are grown overnight in a 1 ml culture, then a fresh10 ml culture re-inoculated the next day when transformation is tooccur. The bacteria grow into log phase, and are harvested at a densityof no more than OD600=0.6 and preferably between 0.2 and 0.5. Thebacteria are then centrifuged to remove the media and resuspended in theco-culture medium. Since Hi II is used, medium preferred for Hi II isused. This medium is described in considerable detail by Armstrong, C.I. and Green C. E. “Establishment and maintenance of friable,embryogenic maize callus and involvement of L-proline” Planta (1985)154:207-214. The resuspension medium is the same as that describedabove. All further Hi II media are as described in Armstrong et al. Theresult is redifferentiation of the plant cells and regeneration into aplant. Redifferentiation is sometimes referred to as dedifferentiation,but the former term more accurately describes the process where the cellbegins with a form and identity, is placed on a medium in which it losesthat identity, and becomes “reprogrammed” to have a new identity. Thusthe scutellum cells become embryogenic callus.

[0076] It is preferred to select the highest level of expression of theenzyme, and it is thus useful to ascertain expression levels intransformed plant cells, transgenic plants and tissue specificexpression. For enzymes, one such detection method is to determine theactivity of the enzyme using a substrate specific for the type ofreaction catalysed by the enzyme. For example, laccase activity can bedetected using any number of colorometric substrates such ABTS(2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) by incubatingsolutions of laccase with the substrate in excess in a buffer at theappropriate pH and monitoring the change in absorbance over time.

[0077] The levels of expression of the gene of interest can be enhancedby the stable maintenance of an enzyme encoding gene on a chromosome ofthe transgenic plant. Use of linked genes, with herbicide resistance inphysical proximity to the enzyme encoding gene, would allow formaintaining selective pressure on the transgenic plant population andfor those plants where the genes of interest are not lost.

[0078] With transgenic plants according to the present invention, enzymecan be produced in commercial quantities. Thus, the selection andpropagation techniques described above yield a plurality of transgenicplants which are harvested in a conventional manner. The plant with theenzyme can be used in the processing, or the enzyme extracted. Enzymeextraction from biomass can be accomplished by known methods which arediscussed, for example, by Heney and Orr (1981).

[0079] It is evident to one skilled in the art that there can be loss ofmaterial in any extraction method used. Thus, a minimum level ofexpression is required for the process to be economically feasible. Forthe relatively small number of transgenic plants that show higher levelsof expression, a genetic map can be generated, via conventional RFLP andPCR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson (1993). Genetic mapping can be effected, first toidentify DNA fragments which contain the integrated DNA and then tolocate the integration site more precisely. This further analysis wouldconsist primarily of DNA hybridizations, subcloning and sequencing. Theinformation thus obtained would allow for the cloning of a correspondingDNA fragment from a plant not engineered with a heterologous enzymeencoding gene. (Here, “corresponding” refers to a DNA fragment thathybridizes under stringent conditions to the fragment containing theenzyme encoding gene). The cloned fragment can be used for high levelexpression of another gene of interest. This is accomplished byintroducing the other gene into the plant chromosome, at a position andin an orientation corresponding to that of the heterologous gene. Theinsertion site for the gene of interest would not necessarily have to beprecisely the same as that of the enzyme encoding gene, but simply innear proximity. Integration of an expression vector constructed asdescribed above, into the plant chromosome then would be accomplishedvia recombination between the cloned plant DNA fragment and thechromosome. Recombinants, where the gene of interest resides on thechromosome in a position corresponding to that of the highly expressedenzyme encoding gene likewise should express the gene at high levels.

[0080] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed.

[0081] While the examples are directed to particular enzymes, it isevident that any enzyme produced in a plant by introduction ofheterologous DNA encoding the enzyme, where that enzyme requires atransitional metal cofactor to be in the active form, is encompassedwithin the scope of the invention.

[0082] The following are presented by way of illustration and are notintended to limit the scope of the invention. The references cited inthis specification are incorporated herein by reference.

EXAMPLE 1 Laccase Extraction with Copper—Background Methodology

[0083] Lignin is a biopolymer of plants that is a major component ofsecondary cell walls (Bonner and Varner, 1976). This complex polymer isformed from oxidized phenolics produced through the action of oxidasessuch as peroxidase or laccase. The use of plant cell wall materials suchas wood, wheat straw or corn stover as a source of fiber, fuel or feedrequires the removal, degradation or modification of lignin. Currently,processes to remove lignin or disrupt and reform lignin bonds aregenerally chemical processes and are highly polluting. Improvedprocesses which lower pollution are being sought.

[0084] In this regard, enzymes secreted from wood rot fungi can beutilized to modify lignin. Laccases are one class of these enzymes (Calland Mucke, 1997), called blue copper oxidases and use copper to acceptand donate electrons in the oxidation and reduction of substrates. Thepresence and oxidation state of copper in these enzymes is critical totheir maximal activity. Laccase activity oxidizes the phenol componentsof the lignin (Solomon et al., 1996; Yaropolov et al., 1994). Thisaction on a large scale can be applied to many industrial processes. Inan effort to produce large amounts of laccases for industrialapplications, the plant expression system is utilized and heretransgenic Zea mays L. is used as a biofactory. The Trametes versicolorlaccase I gene was cloned (Ong et al. 1997) and placed under the controlof maize promoter elements to induce high expression. The source of thegene is not critical to achieving laccase expression, and theStachyboytrys laccase gene as described below was also used in theseexperiments, where indicated. One of the most important factors insuccessful expression of this enzyme in active form in maize is thetransition metal, copper. Without copper one may successfully expressinactive laccase. Copper is important for laccase activation, stablehigh expression in the plant, and enzyme stability in an extract. Theinventors here have discovered that providing additional copper overthat already in the plant is important for obtaining laccase in anactive form.

[0085] Isolation and Cloning of Laccase Encoding DNA

[0086] Attempts have been made to introduce laccase-encoding nucleotidesequences into plants for the purpose of changing the lignin content ofthe plant in WO 98/11205 and WO97/45549. Commercially acceptable levelsof laccase production is taught at WO 00/20615.

[0087] The gene for laccase was cloned from Trametes versicolor by themethods described here, with isolated RNA reverse transcribed into cDNA.The sequence is set forth at FIGS. 1A-C and can also be found at Ong, E.et al. (1997).

[0088] Preparation of Plasmids

[0089] FIGS. 2A-D provides a schematic overview of the process used forproduction of the plasmids. (Note the following abbreviations are used:BAASS refers to the barley alpha amylase signal sequence; FSS refers tofungal signal sequences; KDEL is the sequence targeting expression tothe endoplasmic reticulum; ubi refers to a ubiquitin promoter; pinII isthe terminator; CaMV refers to the 35S cauliflower mosaic virus; moPATis the maize optimized pat selectable marker; and OPH refers to theorganophosphate hydrolase gene, all of which are described herein.) Theplasmids containing the barley alpha amylase signal sequences wereproduced by ligating oligomeric sequences encoding the sequence to the5′ end of the laccase gene, then the entire sequence amplified by PCRand cloned into a The sequencing of individual clones followed andconfirmed the presence of the construct. An individual clone was chosenfor further manipulations. To generate plasmid 7718 (FIG. 3)intermediate vectors with BAASS:: laccase were cut with NcoI and HpaIand ligated into vector 2774, which contains the ubiquitin promoter andPinII terminator. Plant ubiquitin promoters are well known in the art,as evidenced by European patent application no. 0 342 926. The entiretranscription unit was cut from 2774 with NheI and NotI and ligated to3770 containing the 35S promoter with the PAT selectable marker betweenthe left and right borders of the Agrobacterium tumefaciens. For plasmid8908 (FIG. 4) the same procedure was employed, and the ubiquitinpromoter of the 2774 vector removed, substituting the globulin promoter.This is the promoter of the maize globulin-1 gene, described byBelanger, F. C. and Kriz, A. L. at “Molecular Basis for AllelicPolymorphism of the Maize Globulin-i gene” Genetics 129:863-972 (1991).It also can be found as accession number L22344 in the Genebankdatabase. The globulin promoter in p3303 was cut with HindIII and NcoI,and vector 2774 having the ubiquitin, barley alpha amylase, laccase andPinII sequences was cut with the same restriction enzymes. The twopieces were then ligated to create plasmid KB254. While there areseveral approaches possible for preparing the plasmid, in this procedurethe HindIII and NarI site from KB254 was used to cut p7718 andsubstitute the globulin promoter for the ubiquitin promoter in 7718 tocreate p8908.

[0090] For plasmids 7017 and 7699, (FIGS. 5 and 6) containing the KDELsequence, the nucleotides for the amino acids lysine, aspartic acid,glutamic acid and leucine (KDEL) were added to the 3′ end of the laccasegene by PCR amplification using a reverse primer containing the KDELsequence. The entire coding sequence was then put into 2774 containingthe ubiquitin promoter and the PinlI terminator. Following this it wascut with NheI and NotI and ligated to 3770 as described above, togenerate 7017 and 7699.

[0091] Transformation of Maize

[0092] Fresh immature zygotic embryos were harvested from Hi-II maizekernels at 1-2 mm in length. The general methods of Agrobacteriumtransformation were used as described by Japan Tobacco, at lshida asmodified and described, supra. Fresh embryos were treated with 0.5 mllog phase Agrobacterium strains EHA 101 as described above. Bacteriawere grown overnight in a rich medium with kanamycin and spectinomycinto an optical density of 0.5 or greater at 600 nm then re-inoculated ina fresh 10 ml culture. The bacteria were allowed to grow into log phaseand were harvested at no more dense than OD600=0.5. The bacterialculture was pelleted and resuspended in a co-culture medium.

[0093] Individual transformation events were identified when they grewrapidly on the bialaphos-containing medium (3 mg/L). The events wereidentified as follows: LCB is an event generated from plasmid 7017; LCCfrom p7699; and LCG from 8908. Two LCB events, and several LCC and LCGevents were selected. Several plants per transformation event wereregenerated from embryogenic calli as described (Hood et al., 1997) andallowed to flower and set seed in the greenhouse. T1 (first generationtransformed) seed was planted in back-cross nurseries and crossed toelite inbreds to develop high-yielding hybrids with good agronomicqualities. Grain for processing is produced from these lines.

[0094] Extraction of Corn Seed

[0095] Five T₁ seeds were pulverized individually and homogenized witheither 20 mM sodium acetate, pH 5.0 (SA), or 20 mM sodium acetate, pH5.0 containing 0.05% Tween-20 (SAT) for enzyme assay analysis For pooledseed samples, 50 seeds were ground together in a coffee grinder andseparate aliquots were extracted as for individual samples. Extractionwas routinely performed with a 1:2-1:5 ratio of seed tissue to buffer.Extracts were centrifuged for 10 minutes at 20,000× g to pellet celldebris and the supernatant was placed in a fresh tube. Timing and amountof CuSO₄ addition are noted for each individual experiment. Proteinprecipitated by the copper treatments was pelleted by centrifugation for10 minutes at 20,000× g and the supernatant was transferred to a freshtube.

[0096] Determination of Total Soluble Protein.

[0097] Total soluble protein in each extract was determined using themicrotiter assay conditions and reagents from Bio-Rad. With this method,total protein was determined by the Bradford method (Bradford, 1976)using the microassay protocol from Bio-Rad (Hercules, Calif.).Basically, a standard curve of known concentrations of bovine serumalbumin (Sigma P7656) were prepared in extraction buffer. Tenmicroliters of standard or sample are pipetted in duplicate into 96-wellpolystyrene plates and 200 μl of diluted protein assay dye reagent isadded to each sample. The plate was then read at 595 nm and the proteinconcentrations of the unknowns are calculated by comparison to thestandard curve. Samples were quantitated by comparison to a standardcurve of bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) from0.5-6 μg. Laccase microtiter plate activity assay

[0098] One to ten μg of soluble corn protein was added per well of a96-well polystyrene microtiter plate (Costar) containing 140 μl 20 mMsodium acetate pH 5.0 containing 0.05% Tween-20 in each well. Thereactions were initiated with 20 μl of 4.5 mM ABTS substrate (Putter,J., and Becker, R., 1981) and the microtiter plate was incubated at 25°C. The plates were read at 420 nm on a Spectromax 340 (MolecularDevices) at several times, usually one hour and 18-22 hours totalduration depending on the concentration of laccase in the sample.Laccase activity was determined by comparison with known amounts ofpurified recombinant Trametes laccase from Aspergillis (See Table 4).

[0099] Western Analysis

[0100] A Western analysis is a variation of the Southern analysistechnique. With a Southern analysis, DNA is cut with restrictionendonucleases and fractionated on an agarose gel to separate the DNA bymolecular weight and then transferring to nylon membranes. It is thenhybridized with the probe fragment which was radioactively labeled with³²P and washed in an SDS solution. In the Western analysis, instead ofisolating DNA, the protein of interest is extracted and placed on anacrylamide gel. The protein is then blotted onto a membrane andcontacted with a labeling substance. See e.g., Hood et al, “CommercialProduction of Avidin from Transgenic Maize; Characterization ofTransformants, Production, Processing, Extraction and Purification”Molecular Breeding 3:291-306 (1997).

[0101] Laccase samples were analyzed by Western blot. Briefly, proteinswere separated on 4-20% acrylamide gels (Novex) under reducing,denaturing conditions and transferred to Immobilon P PVDF (Millipore).Immunoblots were then blocked with 5% nonfat dried milk in Tris bufferedsaline with 0.05% Tween-20 (TBST), followed by incubation withanti-laccase polyclonal antibodies produced in rabbit. The blots werethen probed with anti-rabbit peroxidase conjugate (Roche BoehringerMannheim) and specific cross-reaction was detected with the enhancedchemiluminescent kit from Amersham. (See FIG. 7).

[0102] Biochemical Characterization of Maize-derived Laccase.

[0103] Expression of laccase was monitored for all events and plantsthat produced seed. Five seeds per plant were individually analyzed forlaccase content. Laccase protein was extracted with other cellularproteins soluble in 20 mM sodium acetate, pH 5.0 with 0.05% Tween-20(SAT). After determination of total soluble protein (TSP), the extractswere analyzed by the laccase activity assay in a 96 well microtiterplate, using ABTS as the substrate (Putter and Becker, 1981). Laccaseamounts were determined by comparison with a known amount ofTrametes-derived standard and expressed as a percent of the protein(Table 4). After extraction in either buffer, some extracts were alsoanalyzed by Western blot and compared to Trametes-derived standards.

[0104] The amount of active laccase in transgenic T₁ seed varied byvector and by the events and lines produced from each vector (Table 4).Individual T1 seed were screened for laccase expression by enzyme assayon non-copper treated extracts (Top half of Table 4). Some extracts werere-screened with copper-treatment (Bottom half of Table 4). The LCBevents recovered from the vector that contained two signal sequences(native fungal and BAASS) and the ER retention signal (KDEL) produced ahigh-expressing seed per line that contained active laccase at 0.065%TSP. The first lines of LCC that were produced (also ER targeted)without added copper in tissue culture (see below), expressed activelaccase at 0.02% TSP in the high seed per line. The LCG events recoveredfrom a vector in which the laccase gene is driven by the maizeglobulin-1 promoter produced lines that were the highest expressing inthis experiment. The laccase was targeted to the cell wall, and the highseed was 0.24% TSP active laccase. (Table 4).

[0105] Seed extracts from several T₂ lines of LCB, LCC and LCG wereanalyzed qualitatively and semi-quantitatively by Western blot (FIG. 7)as well as activity assay. Note the Western detects total laccase,whether active or not where the enzyme activity assay detects the levelof active laccase. The blots were developed with antibodies raised inrabbits whose sera were pre-screened for low levels of cross-reactivitywith corn seed proteins (Hood et al, 1997). The T₂ seed extractscontained two bands that were similar in molecular weight (approximately62 and 65 kDa) to the two major bands visualized in the Trametes control(FIG. 7). The intensity of the bands reflects approximately the amountof laccase loaded. For T₂ seed the amounts of laccase detected in theoriginal extracts by activity assay did not correlate with the amount oflaccase estimated from the Western blots, sometimes by as much as 50fold (Table 4). Extracts were brought to a final concentration of 10 mMCuSO₄ by adding the appropriate volume of 1M CuSO₄ stock prepared indistilled water. Extracts were then mixed and incubated at 50° C. forone hour. The addition of CuSO₄ causes protein to pecipitate (Bell etal., 1983) while leaving laccase in solution and this precipitate wasremoved by centrifugation at 10,000× g for ten minutes before analysisby activity assay. TABLE 4 Event T₁, highest single seed T₂, pooled seedConstruct #'s Enzyme Assay Western Enzyme Assay Western No CopperTreatment, Laccase as % TSP LCB 1&2  0.065 0.06 0.002 0.1 LCC 1-8  0.021NA 0.002 NA 9-24 0.0044 NA 0.0021 0.01 LCG 1&3  0.16 0.2 0.1 3 5-13 0.24NA NA NA Treated with 10 mM CuSO4, 1 h @ 50° C., Laccase as % TSP LCB1&2  NA 0.06 0.14 0.1 LCC 1-8  NA NA 0.015 NA 9-24 0.067 NA 0.029 0.01LCG 1&3  0.8 0.2 1.1 3 5-13 0.68 NA 1.4 NA

[0106] After treatment with 10 mM CuSO₄ for one hour at 50° C., theamount of active laccase detected by activity assay increased 3-10 foldas compared to the untreated samples (see Table 4). This was the casenot only for T₁ seed from many different events and constructs, but forsubsequent generations of seed produced in the field as well. The amountof laccase detected by enzyme assay after treatment with copper sulfatecorrelates much more closely with the amount determined by Westernanalysis (see T₂ data above). Due to protein precipitation, the actualamount of laccase recovered as a perecent of the soluble protein leftafter precipitation can increase as much as ten fold, achieving anywherefrom 10% to 90% enrichment of laccase depending upon the conditions ofthe copper treatment. This results in laccase activity levels that are110-fold higher in the copper treated extract. Therefore, the assayfigures in Table 4 use the protein concentration for an untreatedsample, and show the amount of active laccase after copper treatment,allowing comparison of the percent total soluble protein numbers in theupper and lower parts of the table. Samples on Western blots are notaffected by copper treatment and the concentrations predicted are arough estimate.

[0107] In some events, particularly LCC events, expression level incallus and leaf tissue was also monitored. This was primarily donebecause the first attempts to produce plants from transgenic events fromthis vector were not successful. Though events were recovered at a lowfrequency, as soon as plants were regenerated and placed in the light,the growing point (meristem) died. Consequently, the only event thatsurvived to produce seed was an event that showed quite low expressionof active laccase in seed (0.02% TSP). One possibility for this failurecould be that the presence of laccase in the transgenic events haddetrimental effects. Alternatively, the laccase could more simply beusing up all available copper, and other essential copper-requiringenzymes in the cell were not able to incorporate copper, possibly makingthem inactive. To test the latter hypothesis, 0.025 mg/L copper salt wasadded to the callus selection medium and 23 healthy events wererecovered (data not shown). In assays of the callus material, highlevels of laccase activity were detected in these events selected oncopper.

EXAMPLE 2 Laccase Recovery from Field Samples

[0108] Rows of plants derived from single ears of T₁ seed generated inthe greenhouse were planted in nurseries for back-crossing to eliteinbreds. For the first few lines of LCB, the first generation in thefield (T₂ seed) yielded very low amounts of active laccase whenextracted with SAT and analyzed in the enzyme assay without coppertreatment (Table 5, T₂ seed pools). To examine whether yields could beimproved by external application of copper, the field was sprayed atpollen shed and again two weeks later with Keyplex micronutrients (aliquid fertilizer) containing approximately 0.006% w/v copper ion. Theseed harvested from that field yielded a restored active laccase amount,0.01% TSP (SAT extraction, no copper treatment in vitro), more similarto the T₁ generation seed (Table 5, see T₃ seed). TABLE 5 Copper T₁(high T₂ averaged single exposure seed) seed analysis T₃ pooled seedanalysis Not activated 0.06 0.002 0.0025 Not activated, NA NA 0.01copper sprayed Activated NA 0.035 NA Activated, NA NA 0.024 coppersprayed

[0109] Provision of exogenous foliar copper ions allowed theincorporation of copper ions into the laccase protein enablingapproximately 10 times greater levels to be recovered than those ingrain from the field without added micronutrients or copper. This fieldmaterial showed activation also with copper in vitro, suggesting thatthe affect of copper need not coincide wholly with developmentalaccumulation of the apoprotein. Copper ions are not usually limiting innormal plant development but uptake can be limited on farmlands withhigh soil organic content and pH. Therefore, application of chelatedcopper, a commonly used additive in grain production, to the productionfields to induce consistent accumulation of laccase in grain isfeasible.

[0110] By the process outlined in the above examples, it is possible toachieve a 5-150 fold improvement over initial SAT extracts in the amountof active laccase detected as a percent of total soluble protein fromflour of transgenic seed. These results have implications for thedetection of transgenic protein, its production process and recovery ofthe product. Increased amounts of active laccase are recovered whethercopper is added to the extraction buffer (see Example 3) or after theenzyme has been extracted. Because the amount of copper remaining in thepellet is large when copper is included in the extraction buffer, addingcopper to the extract rather than using it to extract the corn flour isa potentially preferred process, allowing minimal residual copper in theflour.

EXAMPLE 3 Copper in Laccase Recovery

[0111] The following experiments included copper in the extractionbuffer, after extraction and with variable temperatures. Extraction ofcorn seed, determination of soluble protein and laccase microtiter assaywere performed as in Example 1. In addition to SAT (sodium acetatetween), buffers with variations in salt concentration, detergents andreducing agents were used (Table 6). Each sample was extracted twicewith the same buffer and the extracts combined. The buffer containingcopper sulfate (10 mM, #7) had the greatest affect on recovery of activelaccase and reduced the amount of total soluble protein (of all protein)recovered in this experiment by 4.3 fold. This latter result ispartially due to protein precipitation by the copper (Bell et al, 1983).Additionally, 1.6 times more active laccase was recovered in the twoextractions in the copper-containing buffer compared to SAT withoutcopper on a dry weight basis. The result was a solution containing 6.5times more active laccase as a percent of the soluble protein comparedto an SAT extract (Table 6). It was found that copper sulfateselectively precipitated protein from the extract. Surprisingly, laccasewas found in the supernatant. Thus, in addition to increasing the yieldof active laccase from transgenic seed, incubation of the seed extractin copper sulfate containing buffer served to mostly purify that activelaccase. TABLE 6 Extraction of laccase in 8 buffers from LCB pooledseed. Values were determined by enzyme assay. ng lcc/mg mg protein/ LCC% seed in 2 mg seed in TSP in 2 Buffer extracts 2 exts exts #1 20 mMsodium acetate, 0.56 5.2 0.011 0.05% Tween-20 pH 5 #2 50 mM sodiumphosphate, 0.67 5.0 0.013 0.1% sodium lauryl sarcosine, 0.1% TritonX100-pH 7 #3 #2 plus β-Mercaptoethanol- Interference 4.7 X pH 7 #4 #1plus 6.5 mM CHAPS- 0.59 5.4 0.011 pH 5 #5 #1 plus 250 mM ascorbicInterference 4.4 X acid-pH 5 #6 #1 plus protease inhibitor 0.64 6.10.010 cocktail-pH 5 #7 #1 plus 10 mM CuSO₄-pH 5 0.88 1.22 0.072 #8 100mM MES plus 0.05% 0.58 3.7 0.016 Tween-20-pH 7

[0112] To explore whether copper action resulted in the recovery of moreactive laccase or simply improved extraction, LCB flour was extractedwith SAT or SAT plus copper sulfate (10 mM). The SAT extract was broughtto a final concentration of 10 mM copper sulfate subsequent toextraction, and precipitated proteins were pelleted. Laccase enzymeassays were performed on each extract (FIG. 8). Similarly high levels oflaccase were recovered whether the CuSO₄ was added to the extractionbuffer or to the extracted protein, indicating that the CuSO₄ affectsactive laccase recovery and does not improve extraction.

[0113] Substitution for copper with other transition metals was tested(FIG. 9). Three separate extracts of LCB flour were prepared in SAT foreach experimental metal. Each sample was brought to a finalconcentration of 10 mM of the following salts: CuSO₄, FeSO₄, MnSO₄,NiSO₄, and ZnSO₄. The extracts were then incubated at 50° C. for onehour, centrifuged at 10,000× g for 10 minutes and the supernatantsanalyzed for total protein and laccase activity. Shown are the averagesand standard deviations for the three extracts. SAT extracts eitherheated for one hour at 50° C. or not heated were also analyzed ascontrols. The results show copper allows for the recovery of more activelaccase.

[0114] The time and temperature of the CuSO₄ incubation period impactsrecovery of active laccase from LCB seed. LCB corn flour was extractedwith SAT and separated into three aliquots. Each aliquot was brought to10 mM CuSO₄ and incubated at either 4° C., room temperature (about 20°to 27° C.), or 50° C. Each aliquot was sampled at 0, 10, 30, 60, 120,and 180 minutes, precipitated proteins were removed by centrifugationand the laccase activity was determined by enzyme assay. Maximal laccaseactivity was obtained by incubating at 50° C. for one hour or roomtemperature for three hours (FIG. 10). These conditions appear to bespecific to LCB seed either due to the low expression, or perhaps due tothe combination of the KDEL ER targeting sequence and the fungal signalsequence, as set forth more fully in Example 4 below.

EXAMPLE 4 Salt Optimization; Incubation with Chloride Salts

[0115] Chloride is a known inhibitor of laccase activity (Yaropolov etal. 1994). When chloride salts are included in the copper treatmentstep, they can be added separately from the copper sulfate, or used asthe copper salt. In these experiments, the process above was repeated,this time using sodium chloride in addition to the copper sulfate. LCGinstead of LCB seed was used. LCG contains higher laccase expressionlevels (˜10-50 ng/mg seed active laccase after copper treatment). Unlessotherwise noted, corn meal was extracted in 20 mM sodium acetate, pH 5(SA) for one hour at room temperature. Extraction, copper treatments andenzyme assays were performed according to the method outlined inExample 1. Samples containing chloride salts were diluted to less than50 mM (Cl⁻) or dialyzed before analysis of enzyme activity (T. Pohl,1994). For results shown here, samples were diluted about 15 fold intothe assay, resulting in a final chloride concentration of 50 mM or lessin the activity assay. Copper treatment conditions are noted below. Whenonly copper sulfate is used, 14.9 ng/mg active laccase was recovered(FIG. 11). When 0.5 M sodium chloride (final concentration) is added tothe extraction buffer, the amount of laccase recovered with treatmentusing 10 mM copper sulfate (final concentration) increased to 40.8ng/mg. When the sodium chloride is added to the copper sulfate treatmentstep, 40.3 ng/mg laccase is recovered which is almost identical to theamount of laccase obtained when sodium chloride is used in theextraction buffer. (FIG. 11) This indicates that the sodium chloride isenhancing the process by which CuSO₄ restores the laccase activity, notimproving the extraction of more laccase from the seed.

[0116] Addition of copper sulfate alone was compared to addition of 10mM CuS04 (final concentration) with sodium chloride, potassium chloride,sodium sulfate, or sodium acetate (FIG. 12). The presence of thechloride ion is strongly associated with considerable increases inactive laccase recovered. Thus, chloride was found to be an optimalnegative ion that can be added in the incubation with copper. Thechloride salt can be present either during extraction or added after theextract is prepared along with the copper sulfate.

[0117] Optimal sodium chloride salt concentrations are shown to bebetween about 0.2M to 1.5M (final concentration) (FIG. 13). At levelsabove 1.5M, there does not appear to be any additional benefit, althoughno detriment to the process is apparent. Copper concentrations of 10-100mM are optimal for this LCG seed as long as sodium chloride is includedin the copper treatment step. Further, cupric chloride salt can also beused in place of copper sulfate and sodium chloride. (See FIG. 14.) Oneskilled in the art may want to vary the amount of chloride added tomatch the production conditions that are most economically beneficial.Thus, if less chloride is desired, the temperature may be increased ortime of incubation extended.

[0118] As noted above, it is believed that when the negative salt ion isadded to the metal solution, it allows more metal to associate with theenzyme. At the same time, the additional salt appears to make the enzymesomewhat more susceptible to degradation when exposed to highertemperatures Thus, temperature at about 25° to 37° C. is optimum, withroom temperature of about 25° C. to 27° C. the most optimal for at onsetup to 60 minutes. Active laccase recovery with and without salt withvarying copper concentrations, at room temperature and at 50° C. isshown in FIGS. 15A and B. LCB corn extract (FIG. 15A) prepared as inprevious experiments, is incubated at room temperature with either 10 or100 mM copper sulfate over time, with or without 0.5 M sodium chloride.Small aliquots were removed at each time point and centrifuged to removethe precipitated proteins (closed symbols and lines). In addition, analiquot of extract was also incubated at 50° C. for one hour with either10 or 100 mM copper sulfate and either with or without sodium chloride(open symbols). An identical experiment using LCG corn extract wasperformed (FIG. 15B). The addition of sodium chloride allows forconsiderable increases in recovery of active laccase. Further, whenincubated at 50° C., there is a drop in recovery in the results where100 mM CuSO₄ and 0.5 M sodium chloride was used. Thus, the addition ofsodium chloride further enhances active laccase recovery, but should beconducted at lower temperatures, most preferably room temperature up to37° C. Note that the apparent maximal amount of laccase (˜2 ng/mg) wasdetected for LCB with either 10 mM CuSO₄ and 0.5 M NaCl (circles) or 10mM CuSO₄ without 0.5 M NaCl at 50° C. (open square). In contrast, 100 mMCuSO₄ with 0.5 M NaCl, RT gave the apparent maximal amount of laccase(˜5 ng/mg) for the LCG extracts, but incubation of 100 mM CuSO₄ without0.5 M NaCl at 50° C. was actually detrimental to the recovery of activelaccase. Therefore, it appears that either the presence of chloride ionor higher temperatures in the copper treatment step are sufficient toallow for the recovery of laccase from LCB seed, but high temperature inthe copper activation step is not sufficient to allow for maximallaccase recovery from LCG seed.

EXAMPLE 5 Improving Stachybotrys Laccase Recovery

[0119] Background, Vector Construction

[0120] The gene encoding laccase, obtained from the Stachybotrys funguswas expressed in plants, and recovery of active enzyme improved. TheStachybotrys chartarum nucleotide sequence used for this experiment isshown in FIG. 16, and is also described in WO 99/49020.

[0121] To prepare the 8947 plasmid, oligomeric sequences encoding thebarley amylase signal sequence, BAASS, were added to the 5′ end of theStachybotrys laccase gene and the entire sequence up to the PstIrestriction site of the gene was amplified by PCR. The fragment wascloned into a vector backbone resulting in the plasmid K1243. Theaddition of a HpaI restriction site to the 3′ end of the gene was alsoaccomplished with PCR by using a reverse primer containing therestriction site sequence. The resulting plasmid K1222 contains theStachybotrys laccase sequences back to the AscI restriction site of thegene. The plasmid K1272 containing the entire BAASS:Stachybotrys laccasesequences was produced by ligating the HindIII-PstI fragment from K1243,the PstI-AscI fragment from HM642, containing the original StachybotrysLaccase gene, and the AscI-HindIII vector portion of K1222 (FIG. 17A).BAASS:Stachybotrys Laccase contained in the BsmBI-HpaI fragment fromK1272 was ligated into the NcoI-HpaI vector of KB3 81, containing theGlobulin 1 promoter and PinII terminator, resulting in the intermediateplasmid K1369 (FIG. 17A). The entire transcription unit was then cut outof K1369 using HindIII-PmII and ligated into the same sites in 8916resulting in the final plasmid 8947 which contains the 35S promoter withthe PAT selectable marker between the left and right borders of theAgrobacterium tumefaciens (FIG. 17B).

[0122] Treatment of T₂ Seed with Copper

[0123] The highest expressing plants from each of five different eventswas planted in the field. Seed harvested from each of these plants waspooled based on the event from which it was generated. This pooled seedwas ground and two separate extracts were prepared using SA buffer. Theextracts were split into three separate aliquots and either not treated,treated with 10 mM CuSO₄ (final concentration) at 50° C. for one hour,or treated with 30 mM CuSO₄ with 0.5 M NaCl at room temperature for onehour. Table 7 shows the results from this experiment. TABLE 7 CopperTreatment of Stachybotrys laccase Produced in Corn Seed ng activeLaccase/mg Seed Event # No Copper 10 mM CuSO₄, 50° C., 1 h 04 0.02 0.07905 0.039 0.11 09 0.036 0.21 10 0.08 0.74

[0124] Incubation with 10 mM CuSO₄ increases the amount of activelaccase that is recovered from these corn seed extracts. In the bestcase shown here, almost 10 fold more activity was recovered afterincubation with 10 mM CuSO₄ for one hour for event number 10. Sodiumchloride inhibits this enzyme significantly, concentrations as low as100 μM cause 20% inhibition of the activity. While not wishing to bebound by any theory, it is believed that the sodium chloride improvesrecovery of active laccase, however, because of its inhibition while incontact with the laccase, it must be removed before the activationprocess occurs.

[0125] The optimal conditions for incubation were investigated. Event#10 seed was extracted as above and treated with 10 mM CuSO₄ at roomtemperature or 50° C. for 5, 10, 15, 30, 60, and 120 minutes beforeremoving the precipitated proteins and analyzing by enzyme assay. Thelonger incubation times improve the amount of laccase activity recovered(FIG. 18). The higher temperature also improved recovery of laccaseactivity in these relatively low expressing lines.

EXAMPLE 6 Organophosphate Hydrolase Activation with Metals

[0126] Organophosphate Hydrolase (OPH, E.C. 3.1.8.1) is a dimericmetaloenzyme that is capable of breaking down several neurotoxicorganophosphorus compounds. (Di Sioudi, B., 1999). Each dimer has a massof 72,000 Daltons and binds two divalent metal ions per monomer.(Grimsley, J. K, 1997). OPH was first isolated from Pseudomonas diminutaMG and Flavobacterum ATCC 27551. (Di Sioudi, B., 1999). OPH requires thepresence of divalent transition metal co-factors for catalysis and iscapable of using Zn⁺², Mn⁺², Ni⁺², Cd⁺², and Co⁺. (Omburo, G. A., 1992).OPH has previously been successfully expressed in both prokaryotic andeukaryotic systems. Low availability of metal cofactors was observed bythe inventors to be causing the expression of a less active form of theenzyme that can be activated by incubation with the appropriatetransition metal.

[0127] Optimized OPH Gene

[0128] The OPH gene may be obtained from Flavobacterium sp. orPseudomonas diminuta. The amino acid sequence is the same for bothorganisms. The Genbank accession number for the sequence obtained fromFlavobacterium is M29593. The sequence was translated into a proteinsequence and then back translated into a DNA sequence using a maizecodon usage table with the translate and back-translate programs of theGCG Wisconsin package. Wisconsin Package Ver. 9, Genetics Computer Group(Wisconsin). The sequence was altered with the addition of the barleyalpha amylase signal sequence (BAASS). The completed sequence wasanalyzed for unique restriction sites with the Vector NTI program. Fiveroughly equidistant sites were chosen for the construction of the OPHoptimized gene. Oligos were ordered in 50 bp lengths with 25 bpoverhangs. These were annealed and amplified by PCR. Amplified productswere trapped in a vector and transformed into competent cells. Colonieswere analyzed by restriction analysis and by DNA sequencing. Correctclones were then subcloned together in the vector. After the completegene sequence was assembled it was cloned into a Maize expression vectorunder the direction of the ubiquitin promoter and the pinII terminator(p8971 see FIG. 19). The sequence is set forth in FIG. 20.

[0129] Plant Transformation

[0130] Plasmid 8971 was transferred to Agrobacterium by mating asdescribed above. Agrobacterium was used to transform 800 maize embryos.Embryos were transferred to co-cultivation media for 5 days, followed bycounter-selection media for 3 days then the embryos were transferred toselection media and callus was allowed to form. All callus experimentswere done with the first transformant to appear (OPAO 1).

[0131] Callus Preparation

[0132] Callus extracts were prepared by extracting into 20 mM HEPES(N-2-Hydroxyethylpiperazine-N′-2-ethane-sulfonic acid) buffer pH 8.3.Extraction was done using a tissue homogenizer on ice for 2 minutes.Extracts were spun at 10,000× g for 30 minutes. Supernatant was removedand saved and pellets were discarded. Extract was aliquoted and broughtup to 10 mM metal ion with 100 mM solutions of each metal salt (ZnSO₄,NiSO₄, MnSO₄, MgCl₂, and CoCl₂) made up in distilled/deionized (ddi)water. Water (ddi) was added to the No Treatment samples in a volumeequal to that of the metal treatments. Samples were incubated inmicrofuge tubes and temperature was controlled by waterbath (50° and 37°C.) and incubator (25° C.).

[0133] Prep of T₂ Extracts

[0134] Seed derived from OPA event 04 was ground in a coffee grinder.Ground seed was incubated with 3 ml of 20 mM HEPES pH 8.3 per gram ofground seed. Extract was spun for 30 min at 27,000× g. The supernatantwas used for all experiments.

[0135] Incubation

[0136] For those extracts in which salt was added, the extract wasdiluted using 2M NaCl in water. All extracts were brought to the samevolume using water. The no salt treatment was diluted with water alone.Metal was added using IM stock solutions of each metal made up in water.One ml of extract with treatment was transferred to a 1.5 mlmicrocentrifuge tube and incubated at the appropriate temperature (4°C., ˜25° C. (room temperature), 37° C. and 55° C.

[0137] OPH Enzyme Assay

[0138] OPH activity was analyzed by the hydrolysis of paraoxon. Cleavageof paraoxon yields p-Nitrophenol, which is measuredspectrophotometrically at 400 nm. OPH activity was assayed in 1 mlplastic cuvettes by observing the hydrolysis of Paraoxon top-Nitrophenol at 400nm. Units of enzyme were determined using theextinction coefficient of p-Nitrophenol (17 mM⁻¹ cm⁻¹) Each set of datais the average of three assays.

[0139] Negative Ion Addition

[0140] It was previously shown with bacterial enzyme with its metalsremoved that the presence of bicarbonate increased the rate of metalcenter formation. Shim & Raushel, 2000. Corn extracts were prepared asdescribed above and brought to 100 mM bicarbonate with 1M sodiumbicarbonate. The control treatment was diluted with an equal volume ofwater. The samples were then brought to 10 mM CoCl₂ with 1M CoCl₂. Thesamples were then placed in a 37° C. water bath. Enzyme assays wereperformed as described above in 1.5 ml plastic cuvettes with 1 mMParaoxon as the substrate. A total of 5 μl of each sample was used ineach assay and each assay was conducted three times.

[0141] Results

[0142] Enzymatic analysis of extracts made from callus tissue showedvery low activity. However, after incubation with zinc, cobalt, nickelor manganese at 50° C. for 1, hour an increase of up to 50 fold in OPHactivity was recovered when compared to extracted OPH incubated withoutmetal. Incubation with magnesium salt, which is not capable of formingthe active enzyme, showed no activity increase. (FIG. 21A). OPH T₂ seedshowed a similar increase in active enzyme recovered after incubationwith 10 mM cobalt, zinc, nickel or manganese, both chloride and sulfatesalts (FIG. 21B). In both cases, cobalt gave the best overall recoveryof active OPH. Adding 0.1-0.5 M sodium chloride to the incubation bufferdoes not appear to increase the amount of OPH activity recovered and at50° C. actually decreases by 25% the amount recovered. (FIG. 22).

[0143] Increasing the temperature of incubation with the metal increasesthe amount of OPH activity recovered to a point at which the stabilityof the enzyme is compromised (FIG. 23). OPH enzyme increased in activityat all temperatures tested, most slowly at 4° C., reaching maximalactivity after 4 days. Maximal activity was reached at room temperaturein approximately 6 hours, with 37° C. being slightly better at threehours although after 4-5 hours, the enzyme is no longer stable. OPHgains maximal activity most rapidly at 50° C., but activity is reducedafter only 30 minutes. The time and temperature can be manipulated toachieve the best conditions for any given batch of seed.

[0144] Impact of addition of bicarbonate on activation is shown in FIG.24. Recovery of active OPH occurs nearly at the outset of the process,at a higher rate when compared to activation without the negative saltion (FIG. 24).

[0145] OPH expressed in Zea mays shows an increase in activity afterincubation with certain transition metals. The pattern of increase inactivity follows the pattern already shown for substitution in purifiedbacterial enzyme. (Omburo, G. A., 1992). One would not expect to have toadd this cofactor when producing the enzyme in plants, as opposed tobacteria. Bacteria do not contain such transitional metals as a normalpart of their physiology, where a plant has large quantities of themetals, compared to the amount needed to activate the enzyme.

[0146] Thus it can be seen that the invention achieves at least all ofits objectives.

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What is claimed is:
 1. A method of improving recovery of active enzymefrom a plant where the enzyme requires a transitional metal cofactor foractivity comprising introducing into the plant nucleotide sequencesencoding the enzyme and exposing the enzyme to the metal cofactor. 2.The method of claim 1 wherein the transitional metals ions are one ormore of iron, copper, zinc, cobalt, nickel, magnesium, potassium andmanganese.
 3. The method of claim 1 comprising spraying the plant with asolution containing the metal cofactor during plant development.
 4. Themethod of claim 1 comprising extracting the enzyme from the plant with asolution containing the metal cofactor.
 5. The method of claim 1comprising extracting the enzyme from the plant and contacting theenzyme with the metal cofactor.
 6. The method of claim 1 whereinexposure of the enzyme and metal cofactor to recover maximum activeenzyme occurs at a temperature that will not degrade the enzyme.
 7. Themethod of claim 6 further comprising incubating the enzyme and metalcofactor with a negative salt ion.
 8. The method of claim 6 furthercomprising extracting the enzyme prior to exposure to the metalcofactor.
 9. The method of claim 6 wherein the enzyme and metal cofactorare incubated for up to several weeks at a temperature of at least 4° C.and a temperature up to 60° C.
 10. The method of claim 6 wherein theenzyme and metal cofactor are incubated for up to 24 hours at atemperature of at least 18° C. and at a temperature up to 55° C.
 11. Themethod of claim 6 wherein the enzyme and metal cofactor are incubatedfor up to 24 hours at a temperature of at least 20° C. and a temperatureup to 27° C.
 12. The method of claim 6 wherein the enzyme and metalcofactor are incubated for up to three hours at about 50° C.
 13. Themethod of claim 7 wherein the enzyme and metal cofactor are incubatedfor up to 60 minutes at least 18° C. and at a temperature up to 37° C.14. A method of improving recovery of active laccase from a plantcomprising introducing into the plant nucleotide sequences encodinglaccase and exposing the enzyme to copper.
 15. The method of claim 14comprising exposing the plant to the copper by spraying the plant with asolution containing copper during plant development.
 16. The method ofclaim 14 comprising extracting the laccase from the plant and contactingthe laccase with copper.
 17. The method of claim 14 comprisingextracting the laccase from the plant with a solution containing copper.18. The method of claim 14 further comprising extracting the laccasefrom the plant and exposing the laccase to a salt solution duringextraction or after extraction, the salt solution comprising at least0.05 mM copper and comprising no more than 1M copper.
 19. The method ofclaim 18 wherein the salt solution comprises about 1 mM copper andcomprises up to 100 mM copper.
 20. The method of claim 19 wherein thesalt solution comprises at least 10 mM copper and comprises up to 30 mMcopper.
 21. The method of claim 14 comprising incubating the laccase andcopper to recover maximum active enzyme at a temperature that will notdegrade the laccase.
 22. The method of claim 21 comprising extractingthe laccase prior to incubation with the copper.
 23. The method of claim22 comprising incubating the laccase and copper for up to several weeksat a temperature of at least 4° C. and a temperature up to 60° C. 24.The method of claim 23 wherein the laccase and copper are incubated forup to 24 hours at a temperature of at least 18° C. and a temperature upto 55° C.
 25. The method of claim 23 wherein the laccase and copper areincubated for up to 24 hours at a temperature of at least 20° C. and atemperature up to 27° C.
 26. The method of claim 23 wherein the laccaseand copper are incubated for up to three hours at about 50° C.
 27. Themethod of claim 23 wherein the laccase and copper are incubated forabout one hour at about 50° C.
 28. The method of claim 14 comprisingadding a chloride ion salt.
 29. The method of claim 28 wherein thechloride ion salt is sodium or potassium chloride.
 30. The method ofclaim 28 wherein the chloride ion salt is cupric chloride.
 31. Themethod of claim 28 wherein the laccase, copper and chloride salt areincubated for up to several hours at a temperature of at least 18° C.and a temperature up to 37° C.
 32. The method of claim 29 wherein theincubation is about 60 minutes at a temperature of at least 20 and at atemperature up to 27° C.
 33. The method of claim 1 wherein the enzyme isorganophosphate hydrolase.
 34. The method of claim 33 wherein the metalis one or more of the group of zinc, nickel, cobalt or manganese. 35.The method of claim 33 wherein the metal is zinc, nickel, cobalt ormanganese and the metal and extracted organophosphate hydrolase areincubated for at least 15 minutes up to 24 hours at a temperature of atleast 20° and at a temperature up to 27° C.
 36. The method of claim 33comprising adding a bicarbonate ion salt.